The TNF-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine involved in many cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis and inflammation (Wiley S R, Winkles J A (2003) Cytokine Growth Factor Rev 14: 241-249). TWEAK is a type II transmembrane protein that consists of an N-terminal cytoplasmic domain followed by a single transmembrane domain that is separated by a stalk region from the C-terminal Tumor Necrosis Factor (TNF) homology domain (THD) (Chicheportiche Y, Bourdon P R, Xu H, Hsu Y M, Scott H, et al. (1997) J Biol Chem 272: 32401-32410; Feng S L, Guo Y, Factor V M, Thorgeirsson S S, Bell D W, et al. (2000) Am J Pathol 156: 1253-1261). Membrane TWEAK is processed by a protease of the furin family resulting in a soluble ligand containing the THD. The THD functions in ligand trimerization and receptor binding causing TWEAK to signal as a trimerized molecule (Kolzsch J (1990) Z Arztl Fortbild (Jena) 84: 1199-1202; Kolfschoten G M, Pradet-Balade B, Hahne M, Medema J P (2003) Biochem Pharmacol 66: 1427-1432). Importantly, both membrane-bound and soluble TWEAK (sTWEAK) proteins are fully functional and can mediate similar cellular signaling effects by binding to cellular receptors (Brown S A, Ghosh A, Winkles J A (2010) J Biol Chem 285: 17432-17441).
TWEAK acts by binding to the fibroblast growth factor-inducible 14 (Fn14) receptor, the smallest member of the tumor necrosis factor receptor (TNFR) superfamily (Wiley S R, Winkles J A (2003) Cytokine Growth Factor Rev 14: 241-249; Meighan-Mantha R L, Hsu D K, Guo Y, Brown S A, Feng S L, et al. (1999) J Biol Chem 274: 33166-33176). TWEAK-mediated Fn14 signaling triggers a wide range of physiological activities in cells and tissues including blood clotting, cell proliferation, cell migration, inflammation, and angiogenesis (Harada N, et al. (2002) Biochem Biophys Res Commun 299: 488-493; Polek T C, et al. (2003) J Biol Chem 278: 32317-32323). The Fn14 receptor contains a single cysteine-rich domain (CRD) in the extracellular ligand-binding region and a short cytoplasmic tail possessing a single TNFR-associated factor (TRAF) binding site (Wiley S R, Winkles J A (2003) Cytokine Growth Factor Rev 14: 241-249; Meighan-Mantha R L, et al. (1999) J Biol Chem 274: 33166-33176). Notably, TWEAK is the only known TNF superfamily member that can bind to Fn14. Site-directed mutagenesis has demonstrated that TWEAK binding to the Fn14 CRD requires evolutionarily conserved amino acid residues (Asp45, Lys48 and Met50) and all three of the predicted disulfide bonds (Brown S A, Hanscom H N, Vu H, Brew S A, Winkles J A (2006) Biochem J 397: 297-304). Optimal TWEAK-mediated activation of Fn14 is important for promoting productive tissue responses after injury, but excessive TWEAK-Fn14 activation can induce pathological tissue responses, leading to progressive damage and degradation (Burkly L C, Michaelson J S, Zheng T S (2011) Immunol Rev 244: 99-114).
Over-expression of Fn14 has been reported in multiple cancers including glioblastoma, breast, pancreatic, esophageal, lung, and liver carcinomas (Feng S L, Guo Y, Factor V M, Thorgeirsson S S, Bell D W, et al. (2000) Am J Pathol 156: 1253-1261; Han H, Bearss D J, Browne L W, Calaluce R, Nagle R B, et al. (2002) Cancer Res 62: 2890-2896; Tran N L, McDonough W S, Donohue P J, Winkles J A, Berens T J, et al. (2003) Am J Pathol 162: 1313-1321; Watts G S, Tran N L, Berens M E, Bhattacharyya A K, Nelson M A, et al. (2007) Int J Cancer 121: 2132-2139; Willis A L, Tran N L, Chatigny J M, Charlton N, Vu H, et al. (2008) Mol Cancer Res 6: 725-734; Whitsett T G, Cheng E, Inge L, Asrani K, Jameson N M, et al. (2012) Am J Pathol 181: 111-120). In glioblastoma, Fn14 mRNA and protein expression is unregulated in migratory cells in vitro and invading cells in vivo (Tran N L, McDonough W S, Savitch B A, Fortin S P, Winkles J A, et al. (2006) Cancer Res 66: 9535-9542). Fn14 expression increases with increasing tumor grade with highest expression observed in glioblastoma multiforme (Grade IV). In contrast, the expression of Fn14 is minimal to absent in normal brain tissue. Moreover, TWEAK binding to Fn14 triggers glioma cell invasion and survival (Tran N L, McDonough W S, Savitch B A, Fortin S P, Winkles J A, et al. (2006) Cancer Res 66: 9535-9542).
TWEAK-Fn14 signaling plays a key role in various disease states and therefore holds significant therapeutic potential as a novel molecular target for developing anti-cancer and anti-autoimmune therapeutic agents in humans. It has been shown that this interaction plays a pivotal role in various immunological conditions like rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, renal injury, ischaemic stroke, as well as cardiac dysfunction and failure (Burkly L C, et al. (2007) Cytokine 40:1-16; Winkles J A (2008) Nat Rev Drug Discov 7: 411-425; Jain M, Jakubowski A, et al. (2009) Circulation 119: 2058-2068). Several studies have confirmed the therapeutic potential of this pathway in human esophageal and pancreatic cancers, autoimmune disorders (Zheng T S, Burkly L C (2008) J Leukoc Biol 84: 338-347), muscle atrophy and injury (Bhatnagar S, Kumar A (2012) Curr Mol Med 12: 3-13) and chemokine-dependent inflammatory kidney disease (Campbell S, Burkly L C, Gao H X, Berman J W, Su L, et al. (2006) J Immunol 176: 1889-1898). The ever increasing knowledge and data on various downstream reactions stimulated by TWEAK-Fn14 interaction has recently been compiled into a complete repository (Bhattacharjee M, Raju R, Radhakrishnan A, Nanjappa V, Muthusamy B, et al. (2012) J Signal Transduct 2012: 376470). This paves the way for identification of yet unknown components of the signaling pathways.
To date, there are five anti-TNF antibody-based drugs already on the market and 16 out of approximately 22 ligand/receptor pairs under clinical development, constituting one of the most successful classes of biological therapeutics (Tansey M G, Szymkowski D E (2009) Drug Discov Today 14: 1082-1088). These protein-based therapeutics have some notable disadvantages including problems associated with drug delivery, stability, and cost. On the other hand, very few small molecule inhibitors targeting TNFR family members have been identified. Known small molecule inhibitors for the TNFR family act by disrupting trimerization of their respective ligands, as is the case for TNFα (He M M, Smith A S, Oslob J D, Flanagan W M, Braisted A C, et al. (2005) Science 310: 1022-1025) and CD40 (Silvian L F, Friedman J E, Strauch K, Cachero T G, Day E S, et al. (2011) ACS Chem Biol 6: 636-647). Benicchi and coworkers have also focused on the development of a Homogenous Time Resolved Fluorescence (HTRF) assay for identification of small-molecule inhibitors for TWEAK-Fn14 interaction and reported the identification of hits at a rate of 0.007% (Benicchi T, Iozzi S, Svahn A, Axelsson H, Mori E, et al. (2012) J Biomol Screen 17: 933-945). Currently, the potential therapeutic benefit of inhibiting key nodes of the TWEAK-Fn14 signaling pathway remains untapped due to the absence of small molecule tools to interrogate this pathway.
The importance of the Fn14 CRD has been established utilizing an NMR solution structure of this domain and functional mutation studies (Brown S A, Hanscom H N, Vu H, Brew S A, Winkles J A (2006) Biochem J 397: 297-304). The present disclosure is directed to compounds that target the CRD of Fn14 and modulate the TWEAK-Fn14 pathway. The present invention also provides a number of therapeutic uses of these compounds including in the treatment of autoimmune disease.